Abstract

The highly conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs) play a central role in cytoplasmic microtubule organisation and mitotic spindle formation. The fission yeast S. pombe has two family members, Alp14 and Dis1. Both localise to interphase microtubules, spindle pole bodies (the yeast equivalent of the centrosome), and kinetochores. Here we present the characterisation of Alp14 and Dis1 during interphase. We find that Alp14 localisation resembles that of Mal3, a canonical plus end tracking protein. Deletion results in a decrease in the number and length of interphase microtubule bundles at low temperatures. Alp14 is temperature sensitive. At the restrictive temperature we find that an interphasic intranuclear microtubule bundle forms, nucleated from the region of the spindle pole bodies and kinetochores. This intranuclear bundle has a structure and displays dynamics similar to that of a normal interphase bundle and is able to move the nucleus. Dis1 localises to interphase microtubules but does not show plus end tracking behaviour. Deletion has no apparent effect on the organisation of interphase microtubules, but Dis1 is cold sensitive and at the restrictive temperature the cells become blocked in mitosis with aster-like spindles. Deletion of both alp14 and dis1 is lethal. We investigate the functional redundancy between Alp14 and Dis1 during interphase. Over-expression of Dis1 in alp14 deletion cells can partially rescue the mutant microtubule phenotype. Conversely, attenuated expression of Dis1 in an alp14 deletion background results in almost complete loss of interphase microtubules. We conclude that the presence of at least one of the Dis1/XMAP215 homologues is essential for the maintenance of interphase microtubule arrays. Similar to Alp14, Tip1 is a microtubule plus-end tracking protein, homologous to human CLIP170. Together with the EB1 homologue, Mal3, Tip1 spatially regulates microtubule dynamics, ensuring that the cylindrical cell shape of S. pombe is maintained. In the second part of this thesis the characterisation of the protein SPCC736.15 (Toi4), identified in a screen for Tip1-interacting proteins is presented. During interphase, Toi4p-GFP localises to the central regions of the cell cortex. Shortly before mitosis, Toi4p-GFP begins to accumulate at the cell ends. Concurrent with the onset of mitosis, there is exclusion of Toi4p-GFP from the region of the cell cortex where the actomyosin ring forms and the cell subsequently divides. The S. cerevisiae homologue of Toi4p is Pil1p, which is proposed to be the major component of an endocytic organelle termed the eisosome. We tested for such a role for Toi4 in S. pombe, however we detect no link between Toi4 and endocytosis, suggesting that the homologues, although they have a similar localisation pattern, may perform different functions.